Pharmaceutical composition containing IL-10

ABSTRACT

The present invention provides IL-10 or a fragment or a partially modified form thereof for use in promoting the healing of wounds and fibrotic disorders with reduced scarring and methods for same.

[0001] The present invention concerns pharmaceutical compositions forpromoting the healing of wounds or fibrotic disorders, in particular forpromoting the healing of wounds or fibrotic disorders with reducedscarring.

[0002] By “wounds or fibrotic disorders” is meant any condition whichmay result in the formation of scar tissue. In particular, this includesthe healing of skin wounds, the repair of tendon damage, the healing ofcrush injuries, the healing of wounds to the eye, including wounds tothe cornea, the healing of central nervous system (CNS) injuries,conditions which result in the formation of scar tissue in the CNS, scartissue formation resulting from strokes, and tissue adhesion, forexample, as a result of injury or surgery (this may apply to e.g. tendonhealing and abdominal strictures and adhesions). Examples of fibroticdisorders include pulmonary fibrosis, glomerulonephritis, cirrhosis ofthe liver, systemic sclerosis, scleroderma and proliferativevitreoretinopathy.

[0003] By “reduced scarring” is meant reduced level of scarring relativeto an untreated wound or fibrotic disorder.

[0004] In particular, there is a lack of compositions for promoting thehealing of wounds or fibrotic disorders with reduced scarring. Scartissue formation, although providing mechanical strength to a healedwound, can be unsightly and may impair the function of the tissue.

[0005] This is particularly the case in wounds which result in scartissue formation in the CNS, the scar tissue inhibiting the reconnectionof severed or re-growing nerve ends, so significantly affecting theirfunction.

[0006] There is also a lack of compositions for treating and promotingthe healing of chronic wounds, for example venous ulcers, diabeticulcers and bed sores (decubitus ulcers), especially in the elderly andwheel chair bound patients. Such compositions may be extremely useful inpatients where wound healing is either slow or in whom the wound healingprocess has not yet started. Such compositions may be used to“kick-start” wound healing and may then be used in combination withcompositions for promoting healing with reduced scarring. Hence not onlymay a chronic wound be healed, but it may be healed with reducedscarring.

[0007] IL-10 (Interleukin-10) was originally identified as a product ofTh2 cells (Fiorentino, D. F. and Moddman, T. R., 1989, J. Exp. Med.,170: 2081-2095) but was also independently identified (O'Garra, A. etal., 1990, Internal Immunol., 2: 821-823) as a product of B-celllymphomas that prolonged the survival of mast cells and enhancedproliferation of thymocytes.

[0008] Molecular characterisation of human and murine IL- 10 by Moore,K. W. et al. (1990, Science, 248: 1230-1234) and Vieira, P. et al.(1991, Proc. Natl. Acad. Sci. USA, 88: 1172-1176) showed that there wasan 80% homology of their nucleotide sequences. Mouse IL-10 (mIL- 10)protein consists of 157 amino acids with two potential N-glycosylationsites although glycosylation is not essential for the biologicalactivities of mIL-10. Human IL-10 (hIL-10) protein consists of 160 aminoacids with one potential N-glycosylation site which is not used (Vieiraet al., 1991). Both mIL-10 and hIL-10 contain four cysteine residuesthat form two intramolecular disulphide bonds generating biologicallyactive homodimers with molecular weights of 32 kDa and 39 kDarespectively, and it is not clear whether monomeric forms of IL-10 arebiologically active. Although there is 80% homology between hIL-10 andmIL-10, only hIL-10 acts on both human and mouse cells, whereas mIL-10has species specificity activity (Vieira et al., 1991; Kim, J. M. etal., 1992, J. Inmunol., 148: 3618-3623).

[0009] There are many cellular sources and major biological activitiesof IL-10, all of which may play some role in the wound microenvironmentIt has been shown that IL-10 possesses many stimulatory and inhibitoryeffects - van Vlasselar et al. (1994, J. Cell Biol., 124: 569-577)showed that IL-10 inhibited TGF-β synthesis required for osteogeniccommitment of mouse bone marrow cells, and hence the resultingmineralised matrix, whereas Go et al (1990, J. Exp. Med., 172:1625-1631) showed IL-10to be a novel B-cell stimulatory factor. IL-10has also been shown by Bogdan et al. (1991, J. Exp. Med., 174:1549-1555) to directly act on macrophages and inhibit their subsequentactivation and hence release of pro-inflammatory cytokines (see alsoBerg. D. J. et al., 1995, J. Exp. Med., 182: 99-10; Chemoff, A. E. etal., 1995, J. Immunol. 154 (10): 5492-5499).

[0010] Despite the aforementioned studies of cytokines, the presentinventor has found that, surprisingly, IL-10 may be used to promote thehealing of wounds or fibrotic disorders with reduced scarring. Itappears that by inhibiting inflammation at a wound site or site of afibrotic disorder, in particular at an early stage after wounding/onset,there is a “knock-on” effect upon the resulting collagen matrix,resulting in an improved architecture and reduced scarring. This resultis particularly surprising since in the short-term, there was noinhibition of re-epithelialisation or early wound repair, whilst in thelonger-term, it improved the quality of later scar formation and reducedscarring.

[0011] According to the present invention there is provided IL-10 or afragment or a partially modified form thereof for use in promoting thehealing of wounds or fibrotic disorders with reduced scarring.

[0012] By “fragment or partially modified form thereof” is meant afragment or partially modified form of IL-10 which retains theanti-inflammatory healing functionality of IL-10, although. it may ofcourse have additional functionality. Partial modification may, forexample, be by way of addition, deletion or substitution of amino acidresidues. For example, a substitution may be a conserved substitution.Hence the partially modified molecules may be homologues of IL-10. Theymay, for example, have at least 40% homology with IL-10. They may forexample have at least 50, 60, 70, 80, 90 or 95% homology with IL-10.

[0013] IL-10 or a fragment or a partially modified form thereof may befor use in conjunction with a pharmaceutically acceptable carrier,diluent or excipient.

[0014] IL-10 or a fragment or a partially modified form thereof may befor use in conjunction with a composition for promoting the healing ofwounds of fibrotic disorders with reduced scarring.

[0015] IL-10 or a fragment or a partially modified form thereof may befor use in conjunction with a composition for promoting the healing ofchronic wounds.

[0016] Also provided according to the present invention is a method ofpromoting the healing of wounds or fibrotic disorders with reducedscarring comprising the use of IL-10 or a fragment or a partiallymodified form thereof.

[0017] IL-10 or a fragment or a partially modified form thereof may beadministered to a wound site or site of a fibrotic disorder.

[0018] IL-10 or a fragment or a partially modified form thereof may beadministered at a concentration of between about 1 μM and about 10 μM.It may be administered at a concentration of between about 2.5 μM andabout 5 μM.

[0019] IL-10 or a fragment or a partially modified form thereof may beadministered immediately prior to wound healing, but may be effective ifadministered within about 7 days of wounding. It could be administeredon at least two occasions.

[0020] The method may be used in conjunction with a method orcomposition for promoting the healing of wounds or fibrotic disorderswith reduced scarring.

[0021] The method may be used in conjunction with a method orcomposition for promoting the healing of chronic wounds.

[0022] The invention will be further apparent from the followingexample, with reference to the several figures of the accompanyingdrawings, which shows, by way of example only, compositions and methodsof promoting the healing of wounds or fibrotic disorders with reducedscarring.

[0023] Of the figures:

[0024]FIG. 1 shows the inflammatory profile of incisional wounds treatedwith IL-10, injected at day 0;

[0025]FIG. 2 shows the inflammatory profile of incisional wounds treatedwith IL-10, injected at days 0 and 7;

[0026]FIG. 3 shows the blood vessel profile of incisional wounds treatedwith IL-10, injected at day 0; and

[0027]FIG. 4 shows the blood vessel profile of incisional wounds treatedwith IL-10, injected at days 0 and 7.

EXPERIMENTAL

[0028] Rats were wounded and treated with various compositions and thenharvested and the wounds analysed in order to analyse the effects ofanti-inflammatory cytokines upon wound healing. Results show that in theshort-term, there was no inhibition of re-epithelialisation or earlywound repair, whilst in the longer-term, it improves the quality oflater scar formation and reduced scarring.

[0029] Material and Methods

[0030] Male Sprague Dawley rats age and weight matched at 200-250 g wereanaesthetised using equal parts halothane, oxygen and nitrous oxide. 1cm full thickness (through the panniculus carnosus) linear incisionswere made 5 and 8 cm from the base of the skull and 1 cm either side ofthe dorsal midline. The wounds were treated by intradermal injectionwith either 100 μl of IL-10 (2.5 μg/ml) (Genzyme) or phosphate bufferedsaline (PBS) for control. Animals were split into three groups: group Awere injected with IL-10 or PBS on day 0 prior to wounding, group B wereinjected with IL-10 or PBS on day 0 prior to wounding and day 7 postwounding (pw). A third group (C) had the same injection regime as groupB although they were treated with double the dose of IL-10 (5 μg/ml).Animals were killed on days 3 (group A only), 7, 14 and 84 postwounding. Wounds and approximately 0.5 cm of normal skin either side,were excised and bisected for routine wax histology andimmunocytochemistry.

[0031] A further group of eight animals were injected with 100 μl ofIL-10 (1.25 μg/ml) on days 0 and 7 only. Animals were killed on 7 and 84days post wounding. After macroscopic analysis wounds were excised forroutine histology and immunocytochemistry as before. A repeat group ofeight animals were injected with 100 μI of IL-10 (2.5 μg/ml) and killedat 84 days post wounding. After macroscopic analysis wounds were excisedand treated as before. 7 μm-thick wax sections were cut and stained withHaemotoxylin and eosin, Mallory's and Masson's collagen trichrome stainfor the assessment of cellular infiltate and collagen architecturerespectively and Gomori aldehyde fushin stain for elastin. 7 μm-thickcryosections were cut and stained with antibodies to assess inflammation(EDI; Serotec), angiogenesis (von Willebrand factor) and extracellularmatrix deposition (fibronectin and collagen I). Wound sections wereanalysed in detail using a Joyce Lobel image analysis Magiscan. Sixareas, within the wound margins below the epidermal/dermal junction andabove the dermal/panniculus junction, were viewed through a ×10objective and images were captured and using the analysis package GENIAS25 (Joyce Lobel) the area stained within the field was obtained. Resultsare collated and presented as means and standard errors (FIGS. 1 to 4).

[0032] Results

[0033] Macroscopic

[0034] Macroscopic appearances of treated and control wounds werecaptured using a PC image analysis system. The wounds were scored on alinear scale from 0-5 with 0 being normal dermis and 5 a bad scar. 90%of treated wounds score 2 or less, whereas 10% were in the 3 and 4bracket 90% of control wounds scored 3 or more and 10% scored 2 or less.Macroscopically there appears to be less scar formation with treatmentof IL-10 compared to controls.

[0035] Histology

[0036] Qualitative analysis of H&E (Haemotoxylin and eosin) stainedwound sections shows that IL-10 treatment decrease the number ofinflammatory cells influxing into the wound at day 3 and 7 post woundingwhen compared to PBS treatment (control). The degree of scarring isqualitatively assessed by studying Masson's trichrome stained woundsections at 84 days post wounding and grading features of the neodermissuch as fibre size, length and density. Wounds treated with IL-10 (2.5μg/ml) on day 0 only show improved restitution of the dermalarchitecture when compared with control wounds. The IL-10 treated woundshave larger, less densely packed fibres in a more random orientation(basket weave) compared with control wounds where the collagen fibresare finer, more densely packed and aligned parallel to the epidermis.When wounds are treated on day 0 and day 7 with IL-10 (2.5 μg/ml), theresultant dermal architecture resembles normal skin with a more basketweave configuration of the collagen fibres within the wound. Theappearance of the scar is far superior to control wounds and woundstreated with IL-10 on day 0 only. 2.5 μg/ml of IL-10 appears to be themaximal dose as wounds treated with the higher dose (5 μg/ml) have amore visible macroscopic scar. Elastin architecture was assessed usingGomori aldehyde fushin stain. In early control or treated wounds therewas little elastin staining when compared to normal dermis but at 84days although there were fewer fibres in wounds compared to normaldermis there was an increase in elastin staining in IL-10 treated woundscompared to controls. The elastin fibres were associated with thecollagen fibres in the scar. Whilst IL-10 treatment appears to inhibitinflammation and improve the quality of later scar formation, it doesnot inhibit re-epithelialisation or early wound repair.

[0037] Immunocytochemistry

[0038] Qualitative histological analysis was further corroborated byquantitative image analysis which shows that IL-10 inhibits theinfiltration of monocytes and macrophages into the wound when comparedto controls (FIGS. 1 and 2), although IL-10 has no effect onangiogenesis within the wound when compared to controls (FIGS. 3 and 4).Staining for fibronectin shows that IL-10 treated wounds have lessfibronectin present in the wound area at 3 and 7 days when compared withcontrol wounds. Immunostaining for transforming growth factor beta 1(TGFβ₁) showed little differences in cellular staining (mainlymonocytic) between control and treated wounds although there were fewercells in the IL-10 treated wounds.

[0039] Tables 1-4 (below) show the results contained in FIGS. 1-4respectively. Results are given as area stained (μm²)×100, followed bythe standard error of the mean in brackets (n=4). Results given as zeroindicate that there was no detectable staining. TABLE 1 (FIG. 1)Inflammatory cell (ED1) profile of incisional wounds treated with IL-10(injected at day 0) IL-10 Control 3 days pw 15.269 (1.578) 51.004(2.246) 7 days pw 0.321 (0) 71.704 (3.384)

[0040] TABLE 2 (FIG. 2) Inflammatory cell profile of incisional woundstreated with IL-10 (injected at days 0 and 7) IL-10 Control  7 days pw3.123 (1.109) 71.704 (3.384) 14 days pw 0 5.041 (0.697)

[0041] TABLE 3 (FIG. 3) Blood vessel profile of incisional woundstreated with IL-10 (Injected at day 0) IL-10 Control 3 days pw 20.456(1.855) 18.118 (1.700) 7 days pw 1.355 (0.719) 4.368 (0.712) 14 days pw 0 4.432 (0.948)

[0042] TABLE 4 (FIG. 4) Blood vessel profile of incisional woundstreated with IL-10 (injected at days 0 and 7) IL-10 Control  7 days pw5.128 (0.069) 4.368 (0.712) 14 days pw 0 4.432 (0.948)

1. The use of IL-10 or a fragment or a partially modified form thereofin promoting the healing of wounds or fibrotic disorders with reducedscarring.
 2. The use of IL-10 or a fragment or a partially modified formthereof according to claim 1 in conjunction with a pharmaceuticallyacceptable carrier, diluent or excipient.
 3. The use of IL-10 or afragment or a partially modified form thereof according to any one ofthe preceding claims in conjunction with a composition for promoting thehealing of wounds with reduced scarring.
 4. The use of IL-10 or afragment or a partially modified form thereof according to any one ofthe preceding claims in conjunction with a composition for promoting thehealing of chronic wounds.
 5. A method for promoting the healing ofwounds or fibrotic disorders with reduced scarring comprising the use ofIL-10 or a fragment or a partially modified form thereof according toany one of the preceding claims.
 6. A method according to claim 5, IL-10being administered to a wound site or site of a fibrotic disorder.
 7. Amethod according to claim 6, wherein IL-10 or a fragment or a partiallymodified form thereof being administered at a concentration of betweenabout 1 μM and about 10 μM.
 8. A method according to claim 7, IL-10 or afragment or a partially modified form thereof being administered at aconcentration of between about 2.5 μM and about 5 μM.
 9. A methodaccording to any one of claims 5-8 for use in conjunction with a methodfor promoting the healing of wounds or fibrotic disorder with reducedscarring.
 10. A method according to any one of claims 5-9 for use inconjunction with a method for promoting the healing of chronic wounds.